Method for treating tumors

ABSTRACT

A method is provided for treatment of a mammalian patient having a tumor by administering to the patient allogenic donor lymphocytes that have been co-cultured in the presence of the patient-derived lymphocytes under conditions sufficient to alloactivate the donor lymphocytes. It is preferred that the donor lymphocytes be introduced intralesionally. This method is preferred for treatment of glioblastoma in humans.

This application is a continuation of U.S. patent application Ser. No.08/616,880, filed Mar. 15, 1996, now issued as U.S. Pat. No. 5,837,233,which is a continuation-in-part application of U.S. patent applicationSer. No. 08/406,388, filed Mar. 17, 1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for inhibiting tumor cellproliferation. More particularly this invention relates to a method forinhibiting tumor cell proliferation by enhancing the patient's abilityto respond immunologically to the tumor.

2. Description of Related Art

Traditional therapies have done little to alter the outcome for patientswith high-grade brain tumors, such as glioblastomas, and many othertypes of tumors, such as systemic melanoma, and cancers of the head andneck. Patients with resectable primary tumors generally experiencerecurrence of the tumor within one year after surgery, chemotherapy, orradiation. Often these tumors progress rapidly, with or without furtherconventional therapy. Thus, there is the need to develop new modes oftherapy for these deadly tumors.

A new family of cancer therapies developed. in recent years are based onimmunotherapy. In general, tumor immunotherapies take one of twoapproaches: 1) various techniques are employed to activate the patient'simmune system to attack the tumor; or 2) the lymphoid cells of thepatient are removed and activated by in vitro techniques to produceanti-cancer activity, and the activated cells are then systemicallyreintroduced into the patient. The clinical effectiveness of thesevarious types of immunotherapy are being evaluated in patients withdifferent types of cancers. However, one of the major problemsassociated with both of these types of immunotherapy is toxicityobserved when immunotherapeutic agents are administered systemically. Amethod developed to avoid this toxicity is intralesional administrationof immunotherapy, for instance by injection directly into the tumor.Intralesional administration of various forms of immunotherapy to cancerpatients does not cause the toxicity seen with systemic administrationof immunologic agents (M. Fletcher, et al., Lymphokine Res. 6:45, 1987;H. Rabinowich, et al., Cancer Res. 47:173, 1987; S. A. Rosenberg, etal., Science 233:1318, 1989; and G. Pizz, et al., Int. J. Cancer 34:359,1984).

Recent studies indicated that immunization of animals with tumor cellsthat were genetically engineered to secrete different cytokines enhancedthe induction of a therapeutic immune response. The cytokines arebelieved to induce a complex set of reactions including: a) increasedexpression of tumor antigens; b) inflammation and infiltration of thetumor with host lymphoid cells; c) induction of tumor specific immunity;and d) activation of both nonspecific and specific host anti-tumoreffector mechanisms, which destroy the tumor. However, although thistechnique may ultimately prove useful, because it is extremely costlyand time consuming, its application may be limited.

Studies in experimental animals (primarily mice) show that the chronicrelease of cytokines within a tumor may induce a host anti-tumorresponse and tumor regression. Repeated intralesional injection ofcytokines such as Interleukin-2 (IL-2), Tumor Necrosis Factor (TNF) andInterferon-γ (INF-γ) has been shown to cause regression of cutaneoussarcomas (S. P. Creekmore, et al., Resident and Staff Physician34:23-31, 1988; P. Greenberg, et al., Basic and Tumor Immunology (R.Herberman, ed.) p. 302, 1983; E. Grimm et al., Lymphokines, 9:279-311,1984; G. Forni, et al., Lymphokines 14:335-360, 1987). It has also beenshown that injection into a tumor of the animal's tumor cells that havebeen genetically engineered to secrete cytokines such as IL-2, IL-4, TNFand Granulocyte Monocyte Colony Stimulating Factor (GM-CSF) will inducehost anti-tumor immunity (E. Feron, et al., Cell 60, 397-403, 1990; P.Galumbek, et al., Science 254:713-716, 1991; A. Ascher, et al., J. ofImmunol. 146:3227-3234, 1991). These latter results have been obtainedeven in treatment of tumors that were previously thought to benon-immunogenic.

J. M. Redd, et al. (Cancer Immunology and Immunotherapy 34(5):349, 1992)have shown in rats that allogeneic lymphocytes sensitized against donoralloantigens can inhibit tumor formation when co-injected into the brainof a rat with 9L glioblastoma. In a separate study, both normal andalloimmune spleen cells from Wistar rats were injected into established6-day T9 brain tumors in the Fischer rat. Intralesional injection ofnormal Wistar spleen cells from Wistar rats, previously immunizedagainst Fischer alloantigens, cured the tumors in 50% of the Fischerrats. In contrast, untreated animals and non-responders died within 30days. Survivors appeared completely normal and intracranial injectionsof allogeneic cells into normal rats caused no detectable change inbehavior or survival over a three month period. Histopathologicexamination of brains from treated tumor bearing animals revealed: a)mononuclear cell infiltration, massive tumor necrosis beginning at 2 to4 days and total tumor destruction by 15 days; or 2) cellularinfiltration, early tumor destruction and then tumor regrowthprogressing to death of the animal. No damage to normal brain tissue wasevident at any time in these animals. Tumor regressor animals developedsystemic immunity, for they proved totally resistant to intracranialrechallenge with viable tumor. Although these results in rats are ofinterest, their value in reasonably predicting what would be seen in ahighly unrelated species, such as a human, is highly questionable inview of the considerable species diversity which exists, especially withrespect to the immunological response to tumors.

Human glioma patients with localized and surgically accessible tumorsare logical candidates for intralesional immunotherapy. Multiple Phase Istudies in adult patients with gliomas have been reported employingintratumor implants of autologous peripheral blood lymphocytes activatedin vitro with IL-2. While little clinical effect was noted, negativeside effects were few and occurred only when excessive levels of IL-2were co-administered with the cells (K. S. Jacobs, et al., Cancer Res.,47:2101, 1986; R. Merchant, et al., Neurosurgery 23:725, 1988). Studiesof these patients revealed that survival correlated directly with theability of implanted cells to secrete the cytokine TNF. The discovery ofinhibitors for both TNF and IL-1 in the serum or tumor cyst fluid, andin primary cultures of the tumors from these patients suggests the tumorcells surround themselves with agents that block the host anti-tumorresponse. These inhibitors may prevent cytokine activated implantedcells from remaining active in the tumor long enough to cause itsdestruction. This concept is supported by findings in brain tumorstudies in rats. When IL-2 active lymphoid cells were implanted into C6and T9 glioma brain tumors in Wistar and Fischer rats, respectively,histopathologic examination revealed the implanted IL-2 activatedlymphoid cells only remained in the tumor site for 4 to 6 days (W.Carson et al., J. of Immunotherapy 10(2):131-140, 1991).

The mechanisms operative in causing tumor regression in the animalstreated with allogeneic lymphoid cells include a graft vs. host and hostvs. graft reaction in the tumor site. These powerful immunologicreactions may stimulate high levels of endogenous cytokine production inthe tumor, overcome local levels of cytokine inhibitors, and presumablystimulate infiltration, recruitment and activation of both specific andnon-specific host anti-tumor activity. The tumor regressor animals werefound to be resistant to tumor rechallenge. However, it has heretoforebeen unknown whether any treatments based on similar methods wouldachieve similar results in human subjects sufficient to be consideredeffective in the treatment of human tumors.

Therefore, in view of the limitations of the prior art, new and bettermethods for treating mammalian tumors are needed. In particular, newmethods of intratumor immunotherapy are needed for human cancer patientsfor whom regression of an individual solid tumor could prove lifesaving.

SUMMARY OF THE INVENTION

The present invention provides a method for inhibiting tumors inmammals. In the general practice of this invention, the tumor patient'speripheral blood mononuclear cells (PBMCs) are co-cultured in vitro toinduce a mixed lymphocyte cell reaction with healthy lymphocytes derivedfrom a normal donor, preferably an allogeneic donor. Preferably thenormal lymphocytes are from a donor unrelated to the tumor patient, andpreferably the normal lymphocytes are obtained by leukapheresis fromwhole blood. During co-culture the allogenic donor lymphocytes arespecifically activated against the patient's alloantigens. The mixtureof live alloactivated donor and patient lymphocytes produced by mixedlymphocyte culture are referred to herein as "MLCs." The MLC activatedcells produce a mixture of cytokines which have been shown to induce aprimary immune response in vitro. In the treatment of glioblastoma, forinstance, the patient is a human and the alloactivated lymphocytes aresurgically implanted into the patient's brain at the primary tumor site,optionally together with the patient's PBMCs from the co-culture as aMLC mixture, to induce the patient's immune system to attack autologoustumor cells. In the treatment of other types of tumors, the MLCs areinjected into a tumor site, site of metastases, or body cavity, such asthe peritoneum. Optionally, the MLCs are administered peripherally, suchas at a non-primary tumor site, in the treatment of tumors other thanglioblastoma.

In one embodiment of the invention, the alloactivated donor lymphocytesobtained by co-cultivation with patient-derived lymphocytes are isolatedfrom the MLC mixture and administered intralesionally as an implantplaced directly into the tumor of a patient desiring protection againstrecurrence of a tumor, for instance in the proximity of a surgicallydebulked tumor, or desiring treatment of an inoperable tumor.Alternatively, the alloactivated donor lymphocytes can be administeredperipherally in treatment of a tumor, such as at a secondary ormetastatic tumor site. In another embodiment, the allogenic activatedlymphocytes are co-cultivated with patient-derived lymphocytes, and themixture of MLCs is implanted or introduced peripherally as a mixture.

A BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows survival times of glioblastoma patients treated withintratumor implant of MLC cells.

FIG. 2 is a graph showing MRI scans indicating the reduction in tumorsize in nine glioblastoma patients, two patients, one receiving a singleimplant dose of 4×10⁹ MLCs and one receiving a single implant dose of6×10⁹ MLCs, have continued to date to show a progressive reduction intumor mass over periods of 58 and 74 weeks, respectively.

A DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating tumors in mammalsusing an allograft of donor lymphocytes that have been co-cultured invitro in a mixed lymphocyte (MLC) reaction with the patient'smononuclear cells, preferably from peripheral blood, to activate thedonor lymphocytes against antigens associated with the patient's tumor.It is believed that a graft v. host response and a host v. graftresponse to the allograft induce a powerful immunologic reaction in thetumor site that results in cytokine production and tissue destruction.During this reaction the host lymphoid cells identify both the graftlymphoid cells and tissue as foreign and both are inhibited or destroyedby local and systemic immune response. This host reaction occurs notonly at the implant site of the graft lymphoid cells, but may also occurperipherally, such as at a secondary or metastatic tumor site. Inaddition, the implanted, donor lymphocytes immunologically attack thetumor in a graft v. host response. Thus, a strong immunologic reactionin the center of the tumor produces an environment that overcomes thecytokine inhibitors that may be present at the tumor site. The increasein immunologic reaction will inhibit growth, decrease size and/oreradicate established tumors or developing metastases, as well asinhibit recurrence at the site of surgically debulked tumors. Thus, thepresent invention provides a method for potentiating the systemic immuneresponse to the patient's tumor by introducing into the patient a viablepreparation of the alloactivated cells described herein.

Numerous studies have shown that the in vitro environment in a mixedlymphocyte reaction facilitates an active primary immune response toallogeneic and tumor associated antigens (M. Gately, et al, JNCI69:1245, 1982; S. Lee, et al., J. Experimental Medicine 147:912, 1978;J. Zarling, et al., Nature 262:691, 1976). In the present invention, theMLC reaction occurs within the patient's own tumor tissue, therebystimulating the patient to respond against its own tumor. Preferably theco-culture is performed, generally from 1 to 5 days. Since release ofkey cytokines takes place during the early stages of the co-culture, itis preferred that the co-cultured cells be implanted in the early stagesof the co-culture reaction, usually during the first 48 hours of thereaction when cytokine levels are normally highest. This method resultsin release of key cytokines directly into the tumor tissue so that anenvironment is created that is conducive to antigen recognition anddevelopment of cell mediated immunity directed against the antigen.Standard techniques, such as those based on immunoassay, can be used tomeasure the level of various cytokines, including TNF, LT and gammainterferon, present in the MLC culture supernatants. Levels of TNF andLT vary from 50 to 150 units of biologic activity/ml or 500 to 3500pg/ml of supernatant. As a result of the in vitro co-culture, healthyallogeneic lymphoid cells from a donor are specifically activatedagainst the patient's alloantigens.

The implanted cells are generally allowed to come into direct contactwith tumor cells. Thus, as used herein the term "implant" and"implanted" means that the cells are placed into the patient's body as agroup, optionally within a thickening matrix of coagulated serum orother suspension or gel-like substance.

Examples of tumors that can be treated by the method of this inventioninclude the following:

Brain tumors, such as astrocytoma, oligodendroglioma, ependymoma,medulloblastomas, and PNET (Primitive Neural Ectodermal Tumor);

Pancreatic tumors, such as pancreatic ductal adenocarcinomas.

Lung tumors, such as small and large cell adenocarcinomas, squamous cellcarcinoma, and bronchoalveolarcarcinoma;

Colon tumors, such as epithelial adenocarcinoma, and liver metastases ofthese tumors;

Liver tumors, such as hepatoma, and cholangiocarcinoma;

Breast tumors, such as ductal and lobular adenocarcinoma;

Gynecologic tumors, such as squamous and adenocarcinoma of the uterinecervix, and uterine and ovarian epithelial adenocarcinoma;

Prostate tumors, such as prostatic adenocarcinoma;

Bladder tumors, such as transitional, squamous cell carcinoma;

Tumors of the RES System, such as B and T cell lymphoma (nodular anddiffuse), plasmacytoma and acute and chronic leukemia;

Skin tumors, such as malignant melanoma; and

Soft tissue tumors, such as soft tissue sarcoma and leiomyosarcoma.

In one embodiment of the method of this invention, the alloactivateddonor lymphocytes are implanted at the tumor site, optionally togetherwith the patient's PBMCs from the co-culture, to supplement and enhancethe patient's immune system attack upon autologous tumor cells. Forinstance, in one embodiment of the invention alloactivated human donorlymphocytes obtained by co-cultivation with patient-derived PBMCs orcell lysates of PBMCs are administered intralesionally in a human, forinstance as an implant placed directly into the brain of a patientdesiring protection against recurrence of a glioblastoma tumor. Thealloactivated donor lymphocytes can be implanted in the proximity of asurgically debulked tumor, or a tumor treated by irradiation,chemotherapy, or other appropriate techniques. In another preferredembodiment, the allogeneic activated lymphocytes are co-cultivated withpatient-derived lymphocytes and the mixture of co-cultured allogenic andautologous cells, known herein as "the MLCs," is implantedintralesionally.

The typical implant comprises a therapeutic amount of the allostimulateddonor lymphocytes. As used herein the term "a therapeutic amount" meansa sufficient quantity of MLCs or donor allostimulated lymphoid cellsobtained from the mixed lymphocyte culture to inhibit growth, decreasethe size of and/or eradicate established tumors, and/or prevent tumorrecurrence at the site of a tumor that has been surgically debulked ortreated with chemotherapy or irradiation. More generally, a therapeuticamount may vary with the potency of each batch of alloactivated donorcells; the amount required for the desired therapeutic or other effect,the mode of administration, i.e., whether by direct implant into a tumoror body cavity or by peripheral administration, such as intravenously;and the rate of elimination or breakdown of the MLCs by the body onceimplanted or administered. In accordance with conventional prudentformulating practices, a dosage near the lower end of the useful rangemay be employed initially and the dosage increased or decreased asindicated from the observed response, as in the routine procedure of thephysician. In general, however, a unit dosage for direct implantcomprises from about 2×10⁹ to about 6×10⁹ MLCs. For instance, it hasbeen discovered that in the treatment of brain tumors, the upper limitof cells that can be implanted is about 6×10⁹. Alternatively, a unitdosage for peripheral administration usually comprises from about 2×10⁹to about 2×10¹⁰ MLCs.

The invention further comprises a sterile vial or other containerholding a composition comprising a unit dosage of the MLCs. Typically,the vial or container will bear a label sets forth informationconcerning the pharmaceutical use of the composition in treating a tumorin a human, such as FDA approval for use of the composition in thetreatment of a human having one or more of the tumors against which themethod of treatment of the invention is effective as described herein.

Although any known method of obtaining PBMCs from a donor can be used,it is preferred to obtain approximately 150 to 300 ml of leukapheresissuspension containing the donor PBMCs, utilizing techniques ofleukapheresis that are well known in the art for supportive apheresisaccording to the instructions of the manufacturer of the leukapheresisequipment. For instance, leukapheresis can be performed using a Cobe2997, Cobe Spectra® (Lakewood, Colo.), Fenwall CS 3000 (Deerfield,Ill.), or Haemonetics (Braintree, Mass.) blood cell separator.Generally, a flow rate of 40 to 50 ml/min for 2 to 4 hours withlymphocyte yield of 2-4×10⁹ can be used to process a total donor bloodvolume of 7,000 to 12,000 ml to yield 200 to 250 ml of leukapheresissuspension having less than 1 ml of red cells. For example, if a Cobe2997 blood cell separator is used, the centrifuge rate is generallyabout 5×g, the flow rate is up to about 45 ml/min and the collectionrate is no more than or equal to 2.5 ml/min. One skilled in the art willappreciate that the yield of lymphocytes will vary with the donor andthe leukapheresis machine used. For instance, if the donor pre-absolutelymphocyte counts are in the 0.6×10⁹ to 1.0×10⁹ level, as little as 150ml of donor leukapheresis suspension can be drawn.

If desired, the donor cells can be contacted with a stimulatory cytokinesuch as IL-2 to trigger activation of the cells to the patient-derivedantigen during co-culture and to further stimulate lymphocyteproliferation.

Donor PBMCs are obtained from the donor blood fraction, exercising careto avoid rupture of mononuclear cells, for instance by centrifuging theblood fraction containing the mononuclear cells through a cellseparation medium such as Histopaque® 1.077 at 350×g for 7 to 10minutes. Those of skill in the art will know of other PBMC cellseparation techniques that can readily be utilized. Donor blood istypically pre-screened 3-7 days prior to surgery for HIV, Hepatitis A,B, and C, and VDRL.

Sufficient anticoagulant, such as 2% citrate, is added to the donor andpatient blood or blood fraction to prevent coagulation upon withdrawal.Alternative anticoagulants and mixtures thereof known to one of skill inthe art can also be used, such as anti-coagulant citrate dextroseformula A (ACDA) 15 ml/citrate 100 ml; anti-coagulant citrate dextroseformula B (ACDB) 25 ml/citrate 100 ml; or citrate phosphate dextrose(CPD) 14 ml/citrate 100 ml.

Typically, whole blood is withdrawn from the patient to be treatedaccording to the invention using methods known in the art, such as venapuncture. PBMCs are isolated from patient whole blood, usually bycentrifugation through a cell separation medium, such as Histopaque®1.077 (Sigma, St. Louis, Mo.), and are thoroughly washed to free thecells of the clotting factor in the patient's blood.

Samples of both the donor's and patient's blood or blood fraction shouldbe thoroughly tested to ensure sterility before co-culturing of thecells. Typical of the tests for sterility of blood components that canbe conducted by one of skill in the art are those using such growthmedia as thioglycollate broth, tryptic soy broth and Roswell ParkMemorial Institute Tissue Culture Medium (RPMI) with 10%heat-inactivated fetal bovine serum (FBS)(RPMI--10%) and 1% L-glutaminewith no added antibiotics. Sterility tests utilizing the culture ofblood cells in such growth media are illustrated in Example 1 of thisapplication. Alternative sterility tests will be known to those of skillin the art.

Before mixed lymphocyte culture can be performed, typically the PBMCsisolated from whole blood are analyzed to determine the number of livecells per unit volume. This can be performed, for example, by using astain that differentiates between living and dead cells and counting thecells in a Neubauer chamber. Typical stains for this use are trypan blueand Eosin Y dyes, both of which can be used in wet preparations.Alternative stains will be known to those of skill in the art.Generally, the concentration of live cells is standardized by dilutingthe preparation of PBMCs to achieve a predetermined concentration oflive cells per unit volume. Although one skilled in the art may select anumber somewhat higher or lower, it is generally preferred that thenumber of live cells be fixed at a concentration of about 10⁷ cells/mlfor the purposes of conducting the mixed lymphocyte culture. The donorcells are typically cultured at a ratio of 10:1 to 20:1 compared topatient cells.

Standard techniques for conducting mixed lymphocyte culture usingmammalian (i.e., human) cells are well known in the art and areillustrated in Example 1 of this application. See, for instance, CurrentProtocols in Immunology, Ed. J. E. Coligan, et al., John Wiley & Sons,Inc., 1994, Sec. 7.10 and M. Gately, et al., supra; S. Lee, et al.,supra; and J. Zarling, et al., supra, which are incorporated herein byreference in their entirety. To block response of the patient stimulatorcells to the donor responder cells (back stimulation), it is preferredthat the patient cells be irradiated or treated with a DNA bindingagent, such as mitomycin C, before mixture of the cells duringco-culture to reduce or eliminate patient cell proliferative potential,as is well known in the art. In the present invention, donor lymphocyticcells are typically co-cultured in a short term mixed lymphocyte culturewith the patient's PBMCs for a period of at least 48 hours, andpreferably from 1 to 5 days. As a result of the in vitro co-culture,healthy lymphoid cells from the unrelated donor are specificallyactivated against the patient's tumor associated antigens.

Preparations for parenteral or intravenous administration are typicallycontained in a "physiologically compatible carrier." Since the cellsutilized in the practice of this invention are live, a physiologicallycompatible carrier is one that does not impair viability of the cells,i.e., is hypotonic and at physiological pH. Such carriers includesterile aqueous salt solutions, suspensions and emulsions, includingsaline and buffered media, Ringer's dextrose, dextrose and sodiumchloride, and lactated Ringer's solution. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers, such as thosebased on Ringer's dextrose, and the like. For administration bynon-intravenous routes, the carrier can be in the form of clottedplasma, preferably the patient's clotted plasma. Alternatively thecarrier can be a plasma-free, physiologically compatible, biodegradablesolid or semi-solid, such as a gel, suspension or water soluble jelly.Acacia, methylcellulose and other cellulose derivatives, sodium alginateand tragacanth suspensions or gels are suitable for use as carriers inthe practice of this invention, for example, sodiumcarboxymethylcellulose 2.5%, tragacanth 1.25% and guar gum 0.5%.

In a preferred method of implant at a tumor site, the co-culturedlymphocytes are collected from the co-culture supernatant (after atleast 48 hours of co-culture) by centrifugation at the time of surgery.The collected cells are washed two times with injectable saline andre-suspended in platelet free, decalcified plasma obtained from thepatient the previous day. The cells in plasma are transported tosurgery, the plasma is re-calcified by the addition of calcium,preferably in the form of calcium gluconate so that the plasma clots,embedding the cells in an autologous plasma clot. The patient's tumor isthen surgically debulked, and the clot is aseptically minced andimplanted into the tumor site.

The following examples illustrate the manner in which the invention canbe practiced. It is understood, however, that the examples are for thepurpose of illustration and the invention is not to be regarded aslimited to any of the specific materials or conditions therein.

EXAMPLE 1 A. Mixed Lymphocyte Cell (MLC) Culture Procedure 1. Collectionof Responder PBMC from Unrelated Donor

Peripheral blood mononuclear cells (PBMCs) were collected byleukapheresis from normal healthy donors unrelated to the patient.Donors were pre-screened to test for complete blood count (CBC) withdifferential, Hepatitis A, B, and C, VDRL, and HIV-I.

Approximately 150 to 300 ml of leukapheresis suspension containing PBMCwas collected from each donor, using standard blood donation proceduresfor supportive apheresis according to the manufacturers' instructions.The leukapheresis was performed using a Fenwall CS 3000 (Deerfield,Ill.) blood cell separator. A flow rate of 40 to 50 ml/min for 2 to 4hours with lymphocyte yield of 2-4×10⁹ processed a total donor bloodvolume of 7,000 to 12,000 ml to yield 200 to 250 ml of leukapheresissuspension having less than 1 ml of red cells. If a Cobe 2997 blood cellseparator was used, the centrifuge rate was 5×g, the flow rate was up to45 ml/min, and the collection rate was no more than or equal to 2.5ml/min.

However, if donor pre-absolute lymphocyte counts were in the 0.6×10⁹ to1.0×10⁹ range, as little as 150 ml of leukapheresis product was drawn.Hematocrit for the final product was 3.5%. At least one total bloodvolume was processed for 80% efficiency of lymphocyte collection.

The anticoagulant used was either 2% citrate or a citrate/anticoagulantratio of ACDA--15 ml/citrate--100 ml; ACDB--25 ml/citrate--100 ml; orCPD--14 ml/citrate--100 ml. To obtain the utmost product purity, theactual and final product from the cell separator was transported as apure concentrate of cells in autologous plasma. The cells were notwashed, and no albumin was added.

2. Preparation of Donor Cells

The leukapheresis product was transported to the MC Oncology ResearchLaboratory for the production of allogeneic mixed lymphocyte cells(MLCs) for immunotherapy.

Cells were drained from the leukapheresis pack into two or three 250 mlcentrifuge tubes, removing and setting aside 3 ml for sterility tests tobe done during centrifugation. Cell concentrate was diluted withphosphate buffered saline (PBS) and centrifuged for 7 minutes at 2,000rpm. Centrifugation was repeated twice for a total of three times towash the cells free of the clotting factor in the donor's serum.

Three 1 ml aliquots from the 3 ml removed from the leukocyte suspensionwere placed into sterile capped tubes for sterility testing. The first 1ml aliquot was added to thioglycollate medium (Difco, Detroit, Mich.)(30-35° C., 48 hr.); a second 1 ml was added to tryptic soy broth(Difco, Detroit, Mich.) (25-30° C., 48 hr.); and the third 1 ml wasadded to RPMI 1640 (GIBCO, Gaithersburg, Md.) with 10% heat-inactivatedFBS (RPMI--10%) and 1% L-glutamine, but without antibiotics.

Cells were spin washed twice at 150 g for 10 minutes in PBS to removeplatelets. The supernatant was very carefully discarded as cells were ina slurry and not a pellet. Cells were resuspended in AIM V (GIBCO,Gaithersburg, Md.) supplemented with 2% heat inactivated FBS (2% AIM V)to 420 ml, and placed into a T-175 cm² flask.

Patient or donor blood was diluted 1:1 with sterile saline. For cellseparation, 35 ml of cell suspension was carefully layered onto 15 mlHistopaque® 1.077 suspension medium (Sigma, St. Louis, Mo.) in each 50ml tube and centrifuged at 250 g for 45 minutes. Centrifugation wasstarted slowly and gradually increased to full speed. Aftercentrifugation, the interface containing mononuclear cells between theHistopaque® suspension medium and the plasma layer was carefullycollected with a 25 ml sterile pipet, deposited into clean 50 mlcentrifuge tubes, diluted with 2% AIM V Media 1:1, and centrifuged at550 g for 7 to 10 minutes to form a cell pellet. Cells remained aminimum of time in the Histopaque® suspension medium, because it istoxic to the cells.

The supernatant was discarded, the pellet was resuspended in 2% AIM Vand divided into two 50 ml centrifuge tubes to a total volume 40 ml, andcentrifuged at 550 g for 5 minutes. After washing, the supernatant wasdiscarded. The washing step was repeated twice for a total of threetimes. After the last wash, cells in each tube were resuspended in 50 mlof 2% AIM V. Aliquots of 1 ml of the resuspended cells were diluted to aratio of 1:10 in 2% AIM V per tube, then further diluted 1:1 in TrypanBlue (Sigma, St. Louis, Mo.) to distinguish dead from live cells, andthe live cells were counted in a hemocytometer. Cells were set at 2×10⁶/ml with 2% AIM V.

3. Collection of Stimulator PBMC from Tumor Patients.

From 200 to 400 ml of peripheral blood cells were drawn from eachglioblastoma patient by vena puncture and placed into 250 ml centrifugetubes, removing and setting aside 3 ml for sterility tests to be doneduring spinning. Blood cells in the centrifuge tubes were diluted withsaline and centrifuged for 7 minutes at 550 g. Centrifugation wasrepeated twice for a total of three times to wash the cells free of theclotting factor in the patient's serum. Sterility testing was conductedas described above.

Cells were washed twice by centrifugation at 150 g for 10 minutes insaline to remove platelets, the supernatant was very carefullydiscarded, and 420 ml of cells were resuspended in a T-175 cm² flask insaline.

15 ml of Histopaque® 1.077 cell separation medium was added to twelve 50ml centrifuge tubes, and 35 ml of cells suspended in saline were layeredonto the Histopaque® 1.077 in each 50 ml tube. The cell suspensions werespun at 250 g for 45 minutes, starting centrifugation slowly andgradually increasing speed.

After centrifugation, the mononuclear cells at the interface between theHistopaque® cell separation medium and the plasma layer were carefullycollected with a 25 ml sterile pipet into 2 sterile 250 ml centrifugetubes and diluted with 2% AIM-V to a final volume of 250 ml. The dilutedmononuclear cells were centrifuged at 550 g for 7 to 10 minutes. Forwashing, the supernatant was discarded, then the cell pellet wasre-suspended with 2% AIM V and centrifuged at 550 g for 5 minutes. Thewashing step was repeated for a total of three times.

After the last washing step, cells were re-suspended in 50 ml of 2% AIMV, 1 ml of the cell suspension was diluted 1:10 in 2% AIM-V per tube,and the number of viable cells was determined by enumeration in a 1:1 inTrypan Blue as described above.

The above-described procedures for collection of cells and proof ofsterility have been approved by the State of California for GMP forsterility and quality control.

4. Alloactivation of Patient Mononuclear Cells (PBMC) with DonorLeukocytes

The isolated patient PBMCs were re-suspended at 10⁷ cells/ml in AIM-V,50 μg Mitomycin C (Bristol-Mayer Squibb, Princeton, N.J.) were added perml of patient cell suspension, and the suspension of PBMCs was incubatedat 37° C. for one hour to block response of the stimulator cells to theresponder cells (back stimulation). After one hour of incubation, theexcess mitomycin C was washed from the cells by alternate centrifugation(250 g for 5 min), and the cells were resuspended in AIM-V. Aftermitomycin treatment of the patient's PBMCs, the cells were added at a20:1 to 10:1 donor:patient cell ratio to the donor culture (obtained asdescribed above).

For co-culture, the donor and mitomycin C-treated patient PBMCsuspension was placed in a sealed sterile Fenwal tissue culture systemespecially designed for culture of PBMC for reimplantation intopatients. Cells were passed in sealed systems via Fenwal cell transferunits and pumps according to the manufacturers instructions, andcultured in a 37° C. incubator for 48 hours.

5. Sterility Testing of Alloactivated Cells

Two days prior to implantation of the cell suspension, the followingthree sterility tests were performed. 10 ml sterile aliquots wereremoved from each tissue culture bag, placed into sterile capped 15 mlcentrifuge tubes, and centrifuged for 10 minutes at 450 g. In each tube,the pellet was resuspended in 3.0 ml of PBS. A 1 ml aliquot of the cellsuspension was added to each of three sterile capped tubes containing 2ml of thioglycollate broth, tryptic soy broth, or RPMI--10% andincubated for 48 hours. Each cell suspension was examinedmicroscopically prior to implant to detect signs of microbial growth.

On the day of surgery, the cells were centrifuged out of their medium,washed two times with saline and re-suspended in platelet free,decalcified plasma obtained from the patient the previous day. The cellswere transported to the operating room in plasma, then the plasma wasre-calcified by the addition of calcium gluconate so that it clots justbefore implantation into the tumor bed.

The day of surgery a drop of collected cell pellet was again examinedfor sterility under the microscope. Just prior to clotting, a 100 μlaliquot of the cell suspension was added to 2 ml each of RPMI--10%without antibiotics, thioglycollate and tryptic soy broth in a sterilecapped tube. The samples were then incubated for four days aftersurgery, and a running log was kept of this last sterility test.

Cyto-implants produced by the above described method have received FDAapproval for use in human cancer patients (IND-BB 6288).

B. Intralesional Implant of Mixed Lymphocyte Cell Culture

A human Phase I trial was initiated to test the effects of intracranialinjection of alloactivated allogeneic lymphoid cells in patients withrecurrent glioblastoma. A total of 9 patients with recurrent Grade 3 andGrade 4 glioblastoma were entered into this trial. MLCs obtained frommixed lymphocyte culture in Step 3 above were implanted intralesionallyin the debulked tumor at the time of surgery. Three patients were testedat each of three cell doses of 2×10⁹, 4×10⁹, and 6×10⁹ of MLCs. Thepatients were followed clinically and magnetic resonance imaging (MRI)scans were performed at various monthly intervals on each patientfollowing implantation to monitor the progress of the disease.

All patients complained of headache and nausea for 1 to 2 months aftersurgery. Patients receiving the highest cell dose experienced thehighest number of symptoms. Four of the five patients, including allthose in the highest dose and one each in the lower doses, showed a 50%or greater reduction in the area of tumor enhancement in MRI scans overa 1 to 3 month period after the implant. The results of MRI scans areshown in FIG. 1, which measures survival time of glioblastoma patientstreated with intratumor implants of MLC cells. Two patients, onereceiving a single implant dose of 4×10⁹ MLCs and one receiving a singleimplant dose of 6×10⁹ MLCs, have continued to date to show a progressivereduction in tumor mass over periods of 58 and 74 weeks, respectively.These two patients also have shown a marked improvement in Karnofskyscore. The results of MRI scans are shown in FIG. 2, which measures thereduction of tumor volume as a function of time since implant of MLCs.

EXAMPLE 2

Phase I studies have been conducted in two patients with systemicmelanoma. Mixed cultures of patient and donor lymphocytes were preparedas described in Example 1. The patients were treated by intralesionalinjection of 2×10⁹ MLC cells into cutaneous tumors. Neither patientexhibited negative effects. The first patient experienced necrosis anddestruction of the injected cutaneous tumor and inflammation at adistant metastasis. The second patient showed inflammation at theinjected tumor, but no necrosis at the present dosage level. Treatmentof these patients is ongoing.

EXAMPLE 3

A human Phase I clinical study has been conducted to test the effect ofintratumor implant of MLC cells in patients with pancreatic cancer. Fourpatients with incurable, untreatable pancreatic cancer receivedintralesional implant of 4×10⁹ MLC cells . Little or no toxicity wasfound. A reduction in tumor mass occurred and extension of life span hasbeen observed. Three patients have experienced a greater than 50 percentreduction in tumor mass. In patient No. 3, the serum levels of CA-19-9,a marker for this tumor, declined from a high of 206 to 86 over a twomonth period. Seven months after treatment, two of the treated patientshave returned to their jobs, have no clinical symptoms of tumor, and areliving a totally normal life.

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It should be understood that variousmodifications can be made without departing from the spirit and scope ofthe invention. Accordingly, the following claims are intended to beinterpreted to embrace all such modifications.

What is claimed is:
 1. A method of treating a human patient having atumor, comprising implanting at or around the site of a solid tumor insaid patient a cell population comprising alloactivated human donorlymphocytes, wherein said alloactivated human donor lymphocytes areproduced by coculturing human donor lymphocytes ex vivo with leukocytesfrom said human patient, wherein the tumor is selected from the groupconsisting of melanoma, pancreatic cancer, liver cancer, colon cancer,prostate cancer, and breast cancer, and whereby said patient is treated.2. The method of claim 1, wherein the treatment is effective ininhibiting tumor growth.
 3. The method of claim 1, wherein the treatmentis effective in improving survival time.
 4. The method of claim 1,wherein the tumor is a melanoma.
 5. The method of claim 1, wherein thetumor is a pancreatic cancer.
 6. The method of claim 1, wherein thetumor is a pancreatic ductal adenocarcinoma.
 7. The method of claim 1,wherein the tumor is a liver cancer.
 8. The method of claim 7, whereinthe tumor is a hepatoma.
 9. The method of claim 1, wherein the tumor isa colon cancer.
 10. The method of claim 7, wherein the tumor is a livermetastasis.
 11. The method of claim 1, wherein the tumor is a prostatecancer.
 12. A method for preparing a pharmaceutical compositioncontaining alloactivated human donor lymphocytes for the treatment ofcancer, comprising the steps of:a) obtaining leukocytes from a humanpatient having a tumor selected from the group consisting of melanoma,pancreatic cancer, liver cancer, colon cancer, prostate cancer, andbreast cancer; b) coculturing human donor lymphocytes ex vivo with theleukocytes so as to alloactivate the donor lymphocytes; and c) preparingthe cocultured cells for human administration at a time when thecocultured cells, upon implantation at or around the site of a solidtumor in the patient, are effective in the treatment of the tumor.
 13. Apharmaceutical composition prepared according to the method of claim 12.14. The composition of claim 13, wherein the pharmaceutical compositioncomprises about 2×10⁹ to about 2×10¹⁰ of the cocultured cells.
 15. Amethod for preparing a pharmaceutical composition containingalloactivated human donor lymphocytes for the treatment of cancer,comprising the step of coculturing human donor lymphocytes ex vivo withleukocytes from a patient having a tumor so as to alloactivate the donorlymphocytes, and harvesting the cells from culture at about the timewhen gamma interferon (γ-IFN) secretion by the cultured cells ishighest, wherein the pharmaceutical composition, upon implantation at oraround the site of a solid tumor in the patient is effective in thetreatment of the tumor.
 16. The method of claim 15, wherein theharvesting is at a time which is about 48 hours after initiation of theculture.
 17. A pharmaceutical composition prepared according to themethod of claim
 15. 18. A pharmaceutical composition prepared accordingto the method of claim
 16. 19. The pharmaceutical composition of claim17, wherein the pharmaceutical composition comprises about 2×10⁹ toabout 2×10¹⁰ of the cocultured cells.
 20. A method of treating a humanpatient having a tumor, comprising implanting at or around the site of asolid tumor in the patient a pharmaceutical composition according toclaim
 17. 21. A method for inducing a specific immune response against atumor in a human patient, comprising implanting at or around the site ofa solid tumor in the patient a pharmaceutical composition according toclaim 17.